Production of Transgenic Pig Haboring Cdna of Human Factor Ix Gene (Abstract)

Abstract

Milk protein genes are expressed specifically in the mammary gland. The purpose of this study was to generate transgenic mice and piglets by microinjection of aLA-hFIX gene, a 4.9 kb fragment contained the regulatory sequences of bovineα-lactoalbumin (aLA) and the structure sequence of cDNA from the human factor IX, into the pronucleus of newly fertilized mouse and porcine eggs. This approach is of great advantage in the production of hFIX as a pharmaceutical product in the milk of transgenic animals. A total of 549 embryos, after injection of aLA-hFIX gene, were transfer into fallopian tubes of 18 recipient mice which resulted in 14 (77.8%) becoming pregnancy and 69 pups were obtained at parturition. Fourteen out of 69 (20.3%) pups were confirmed to be transgenic when the genomic DNA from their muscle tissues had been subjected to PCR and Southern analysis. Of the 14 transgenic mice, 3 died soon after birth. Eleven are now sexually matured. The only two transgenic females are found to be infertile after mating with several fertile males. Whereas, all of 9 aLA-hFIX male transgenic founder mice are fertile and the transgene has been found to be germline-transmitted to between 7.1 and 73.3% of the F1 progenies obtained. Western-blot analysis of milk samples obtained from two of the F1 transgenic families has further shown that the milk contains hFIX in a concentration ranged of 75~225μg/ml. Further attempts were then made to generate transgenic piglets carrying the aLA-hFIX. A total of 212 pig embryos after gene injection were also transferred into the fallopian tubes of 11 recipient gilts resulting in three of them becoming pregnant and 14 piglets were obtained from the recipient gilts. Two (1♀ /1♂) of the newborn piglets were confirmed o be as transgenics, according the results from PCR and Southern-blot analysis. Foreign gene of transgenic founder pigs was transmitted to F1 generation progenies with transmission rate ranged between 66.7% and 13.2%. Expression of hFIX cDNA in mammary gland was confirmed by Western-blot analysis of milk samples from the transgenic sow, and the concentration of hFIX contained in the milk was ranged between 200~500μg/ml, with a higher concentration of hFIX found in the milk of later lactation period.